Spectrophotometry is a technique used to measure the amount of light absorbed or transmitted. reflected, and/or emitted by a substance as a function of its wavelength, concentration of the sample, and path length or distance the light traverses through the substance.¹ It is widely employed in chemistry and biology for quantitative analysis of substances, such as determining concentrations of solutes in a solution. Spectrophotometers measure the intensity of transmitted, reflected and/or emitted light at different wavelengths, producing a spectrum that can reveal information about the substance's properties.

Spectrophotometry is used in chemistry for quantitative analysis and in biochemistry for studying enzyme kinetics. It plays a crucial role in environmental testing, detecting pollutants in water and air. In pharmaceutical and medical research, it ensures drug quality and purity and aids diagnostics by supporting biomarker discovery and quantification. In industry, it assesses fuel quality and monitors environmental compliance. Additionally, in material science and semiconductors, spectrophotometry contributes to colorimetry, thin film characterization, and determining magnetic material concentrations.

Principles of Spectrophotometry

Beer-Lambert Law and its role in spectrophotometry

Beer-Lambert law describes the relationship between the absorbance of light by a substance, the concentration of the substance, and the path length of the light through the sample.² In spectrophotometry, this law plays a fundamental role in quantifying the concentration of a solute in a solution. The law is expressed as

A = εcl

In the above expression, A refers to the absorbance. The extinction coefficient or molar absorptivity is ε, the concentration is c, and the path length is l.

This law enables accurate determination of concentration by measuring absorbance, which is crucial in various analytical techniques.

Basic Mechanism of Analysis

Light and matter interaction

In spectrophotometry, light interacts with matter as it passes through a sample.¹ The amount of light absorbed, emitted, or transmitted is measured at different wavelengths, providing valuable information about the substance's concentration and characteristics. This technique is employed in scientific research, particularly in fields like chemistry and biology.

Absorption and transmission of light by a sample

In spectrophotometry, the absorption of light by a sample occurs when specific molecules within the sample absorb photons at particular wavelengths.¹ The transmitted light, on the other hand, represents the portion that passes through the sample without being absorbed. In specific cases, absorbed light is emitted back by the substance, usually at different wavelengths, to produce a Fluorescence spectrum. When emitted light occurs with a time lag, it is generally referred to as Phosphorescence. By measuring the absorption and transmission or emission of light at various wavelengths, spectrophotometry enables the quantification of substances in the sample based on their unique light-interacting characteristics

Types of spectrophotometers

Spectrophotometers come in different types to suit various applications:

• A single-beam spectrophotometer employs a lone light beam for both reference and sample measurements.⁷
• A double-beam spectrophotometer utilizes two separate beams to enhance accuracy.⁸
• UV spectrophotometers are designed specifically for ultraviolet light analysis and are instrumental in studying molecules with UV-absorbing functional groups.⁹
• An IR spectrophotometer measures the absorption or transmission of infrared light by a substance, providing valuable insights into the molecular vibrations and structural characteristics of materials.¹⁰

Featured Product

Advantages of Spectrophotometry

High Sensitivity and Accuracy

Spectrophotometers are highly sensitive instruments capable of detecting minute changes in the absorbance or transmission of light.¹ This high sensitivity allows for the accurate quantification of analytes even at low concentrations. The ability to accurately measure absorbance or transmittance at specific wavelengths enhances precision in determining the concentration of a substance in a sample.

Non-Destructive Nature of Measurement

Spectrophotometric measurements are non-destructive, meaning that the sample being analyzed remains unchanged after the measurement is taken.¹² This is particularly advantageous when working with precious or limited samples. Non-destructive measurements also allow for the continuous monitoring of reactions or processes over time without altering the integrity of the sample.

Spectrophotometry Applications

A spectrophotometer is a versatile analytical tool capable of detecting and quantifying the concentration of substances, identifying impurities, elucidating the structure of organic compounds, monitoring dissolved oxygen levels in aquatic environments, characterizing proteins, detecting functional groups, analyzing respiratory gases in hospital settings, determining the molecular weight of compounds, and for assessing cell viability and determining cell concentration in cell line development.¹¹ Its broad applicability extends to both pure compounds and complex biological preparations, making it an invaluable method for diverse scientific and industrial applications.

Spectrophotometry in Science and Research

Use of spectrophotometry in chemistry and biochemistry

Spectrophotometry is extensively used in chemistry and biochemistry for the quantitative analysis of various substances.¹³ It allows researchers to determine concentrations of analytes by measuring the absorption or transmission of light at specific wavelengths. Biochemical reactions, especially enzyme-catalyzed reactions, often involve changes in absorbance over time. Spectrophotometry is crucial for studying enzyme kinetics, determining reaction rates, and understanding the mechanisms of biochemical processes.¹⁴,¹⁵

Use of spectrophotometry in environmental testing

Spectrophotometry is vital in environmental testing, particularly in the analysis of water quality.¹⁶ It enables the detection and quantification of pollutants, such as heavy metals, organic compounds, and nutrients, contributing to the assessment of environmental impact. Spectrophotometric methods are employed in the analysis of air pollutants.¹⁷ This includes measuring concentrations of gases like ozone, sulfur dioxide, and nitrogen dioxide, which are crucial for assessing air quality and compliance with environmental regulations.

Applications of spectrophotometry in pharmaceutical and medical research

Spectrophotometry plays a key role in pharmaceutical research by aiding in drug development and quality control.¹⁸ It is used to assess the purity, stability, and concentration of drugs, ensuring their effectiveness and safety.

Spectrophotometric assays are utilized in clinical laboratories for diagnostic purposes.¹⁹ For example, measuring the concentration of specific biomarkers or metabolites in blood or urine can provide insights into the health status of an individual.

Spectrophotometry in Industry

Use of spectrophotometry in petroleum and chemical industry

Spectrophotometry is crucial in the petroleum industry for assessing the quality of fuels.²⁰ It enables the measurement of parameters such as octane and cetane numbers, as well as the identification and quantification of impurities. The petroleum and chemical industries employ spectrophotometry for environmental monitoring and compliance. It aids in the detection of pollutants in wastewater and emissions, helping companies adhere to environmental regulations

Use of spectrophotometry in the material science and semiconductor industry

In the field of materials science, spectrophotometry finds application in colorimetry, assessing the radiance of fluorescent printing materials, and monitoring the photodegradation of dyes on transparent films.²¹,²² It plays a crucial role in characterizing thin films, ensuring quality control in nanofabrication processes, and contributing to the engineering of nanoelectromechanical systems. Additionally, spectrophotometry is employed to determine the concentration of magnetic materials in magnetic fluids, underscoring its significance in this scientific discipline. Within the semiconductor industry, spectrophotometry is indispensable for measuring trace anionic contaminants in pure water samples, a critical aspect in industrial processes like semiconductor production and power plant operations.²³ Furthermore, it is applied to characterize thin films used in data storage, semiconductors, and flat panel display devices.²⁴

Developments in Spectrophotometry

Advancements in technology and automation

Automation has markedly progressed spectrophotometry technology, notably through the introduction of automated spectrophotometry (AS).²⁵ AS allows the simultaneous assessment of multiple samples, resulting in a significant boost in laboratory efficiency. This technological advancement has also made continuous monitoring of physiological parameters, such as pulse oximetry, feasible, fostering the development of cost-effective and non-intrusive technologies.²⁶ Furthermore, the refinement of spectrophotometric techniques through advanced technology has elevated their precision, sensitivity, user-friendliness, speed, safety, and applicability.²⁷

Potential for miniaturization and portability

Advances in miniaturization techniques may lead to the development of smaller and more portable “miniaturized” spectrophotometers.²⁸ This is particularly significant for on-site and field applications, allowing researchers and technicians to perform analyses in remote locations without the need for large and stationary equipment. Incidentally, advances in automation have also resulted in the request for miniaturized and integrateable spectrophotometers.

Integration with other analytical techniques

The integration of spectrophotometry with other techniques, known as hyphenated techniques, may become more common.²⁹ This could lead to improved selectivity, sensitivity, and overall analytical power by combining the strengths of different methods.

Impacts on various industries

Spectrophotometry holds considerable promise across diverse industries. Advanced spectrophotometric techniques contribute to the pharmaceutical and medical sectors in the development of personalized medicine and enhancing drug manufacturing processes. In environmental monitoring, real-time analysis capabilities can lead to more effective pollution control measures and resource conservation. The potential for miniaturization and integration with other analytical techniques can bring about significant improvements in areas such as semiconductor manufacturing, materials science, and food safety testing, fostering innovation and efficiency in these industries.

References

1. Beckman AO, Gallaway WS, Kaye W, Ulrich WF. History of spectrophotometry at Beckman instruments, Inc. Analytical chemistry. 1977 Mar 1;49(3):280A-300A.
2. Delgado R. Misuse of Beer–Lambert Law and other calibration curves. Royal Society open science. 2022 Feb 2;9(2):211103.
3. Robinson JW. Atomic absorption spectroscopy. Analytical Chemistry. 1960 Jul 1;32(8):17A-29A.
4. Bings NH, Bogaerts A, Broekaert JA. Atomic spectroscopy. Analytical Chemistry. 2006 Jun 15;78(12):3917-46.
5. Kasha M, McGlynn SP. Molecular electronic spectroscopy. Annual Review of Physical Chemistry. 1956 Oct;7(1):403-24.
6. Duncan JS. Magnetic resonance spectroscopy. Epilepsia. 1996 Jul;37(7):598-605.
7. Schmidt W. A computerized single-beam spectrophotometer: an easy setup. Analytical Biochemistry. 1982 Sep 1;125(1):162-7.
8. Porro TJ, Terhaar DA. Double-beam fluorescence spectrophotometry. Analytical Chemistry. 1976 Nov 1;48(13):1103A-7A.
9. Sharpe MR. Stray light in UV-VIS spectrophotometers. Analytical Chemistry. 1984 Feb 1;56(2):339A-56A.
10. Whetsel KB. Near-infrared spectrophotometry. Applied Spectroscopy Reviews. 1968 Jan 1;2(1):1-67.
11. Karpińska J. Derivative spectrophotometry—recent applications and directions of developments. Talanta. 2004 Nov 15;64(4):801-22.
12. Das AJ, Wahi A, Kothari I, Raskar R. Ultra-portable, wireless smartphone spectrometer for rapid, non-destructive testing of fruit ripeness. Scientific reports. 2016 Sep 8;6(1):32504.
13. Dixit L, Ram S. Quantitative analysis by derivative electronic spectroscopy. Applied Spectroscopy Reviews. 1985 Dec 1;21(4):311-418.
14. Todd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?. Analytical biochemistry. 2001 Sep 15;296(2):179-87.
15. Bender ML, Schonbaum GR, Zerner B. Spectrophotometric Investigations of the Mechanism of α-Chymotrypsin-catalyzed Hydrolyses. Detection of the Acyl-enzyme Intermediate 1-3. Journal of the American Chemical Society. 1962 Jul;84(13):2540-50.
16. Shi Z, Chow CW, Fabris R, Liu J, Jin B. Applications of online UV-Vis spectrophotometer for drinking water quality monitoring and process control: a review. Sensors. 2022 Apr 13;22(8):2987.
17. Scaringelli FP, Saltzman BE, Frey SA. Spectrophotometric determination of atmospheric sulfur dioxide. Analytical Chemistry. 1967 Dec 1;39(14):1709-19.
18. Motwani SK, Chopra S, Ahmad FJ, Khar RK. Validated spectrophotometric methods for the estimation of moxifloxacin in bulk and pharmaceutical formulations. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2007 Oct 1;68(2):250-6.
19. Veskoukis AS, Margaritelis NV, Kyparos A, Paschalis V, Nikolaidis MG. Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network. Redox Report. 2018 Jan 1;23(1):47-56.
20. Korn MD, dos Santos DS, Welz B, Vale MG, Teixeira AP, de Castro Lima D, Ferreira SL. Atomic spectrometric methods for the determination of metals and metalloids in automotive fuels–a review. Talanta. 2007 Aug 15;73(1):1-1.
21. Tominaga S, Hirai K, Horiuchi T. Estimation of fluorescent Donaldson matrices using a spectral imaging system. Optics express. 2018 Jan 22;26(2):2132-48.
22. Stathatos E, Petrova T, Lianos P. Study of the efficiency of visible-light photocatalytic degradation of basic blue adsorbed on pure and doped mesoporous titania films. Langmuir. 2001 Aug 7;17(16):5025-30.
23. Nojavan S, Bidarmanesh T, Memarzadeh F, Chalavi S. Electro‐driven extraction of inorganic anions from water samples and water miscible organic solvents and analysis by ion chromatography. Electrophoresis. 2014 Sep;35(17):2446-53.
24. Ali GA, Emam-Ismail M, El-Hagary M, Shaaban ER, Moustafa SH, Amer MI, Shaban H. Optical and microstructural characterization of nanocrystalline Cu doped ZnO diluted magnetic semiconductor thin film for optoelectronic applications. Optical Materials. 2021 Sep 1;119:111312.
25. Lin K, Xu J, Dong X, Huo Y, Yuan D, Lin H, Zhang Y. An automated spectrophotometric method for the direct determination of nitrite and nitrate in seawater: Nitrite removal with sulfamic acid before nitrate reduction using the vanadium reduction method. Microchemical Journal. 2020 Nov 1;158:105272.
26. Pi S, Camino A, Cepurna W, Wei X, Zhang M, Huang D, Morrison J, Jia Y. Automated spectroscopic retinal oximetry with visible-light optical coherence tomography. Biomedical optics express. 2018 May 1;9(5):2056-67.
27. Tuzson B, Graf M, Ravelid J, Scheidegger P, Kupferschmid A, Looser H, Morales RP, Emmenegger L. A compact QCL spectrometer for mobile, high-precision methane sensing aboard drones. Atmospheric Measurement Techniques. 2020 Sep 7;13(9):4715-26.
28. Koohkan R, Kaykhaii M, Sasani M, Paull B. Fabrication of a smartphone-based spectrophotometer and its application in monitoring concentrations of organic dyes. ACS omega. 2020 Nov 19;5(48):31450-5.
29. Joshi AA, Nerkar PP. Determination of proton pump inhibitors by spectrophotometric, chromatographic and by hyphenated techniques: A review. Critical Reviews in Analytical Chemistry. 2021 Aug 18;51(6):527-48.

recent-articles